OF SPECIFIC IMMUNOTHERAPY
C . Pereira (1), F Botelho (2), B . Tavares (1), C . Lourenco (3),
C . Baeta (3), A .-G . Palma-Carlos (4), J . Lima (3), C . Chieira (1)
SUMMARY: Specific immunotherapy (SIT) is frequently used in the treatment of allergic diseases . However, the mechanisms by which SIT achieves clinical improvement remained unclear. We decided to study the in vivo kinetics of this therapy, using a nuclear medicine approach (leukocytes labelled with 99mTc-HMPAO) in patients on maintenance doses of specific immu-notherapy with confirmed clinical efficacy .
Material and methods : We studied 13 allergic patients grouped according to different treatment schedules : subcutaneous aqueous allergenic extract (3 latex and 2 hymenoptera venom), subcutaneous depot extract (2 house dust mite and 2 pollens), subcutaneous modified allergens (2 pollens), sublingual extract (2 house dust mites) .The control group included two allergic patients submitted to subcutaneous injections of bacterial extract (1 patient - positive control), and aqueous solution (1 patient) . At the same time that the therapeutic allergen was administered subcutaneously, the autologous labelled white cells were injected intravenously in a peripheral vein in the contralateral arm . A thoracic dynamic acquisition of 60 mins, 64x64 matrix, 2 frame/min, in anterior
view
was performed . Static acquisition for 256x256 matrix, during 5 mins each at 60, 90, 120, 180, 240, 300 and 360 mins after the administration of the radiolabelled leukocytes, in thoracic (anterior and posterior), and abdominal view were performed . During the examination, the local erythema was monitored . A similar procedure was undertaken for sublingual administration of immunotherapy .Results : The inflammatory activity at the site of SIT injection (aqueous depot extract) started in the first hour and the increa-se was time related .Formodified allergen extract and sublingual SIT the activity was present since the beginning of the admi-nistration . The ascendant lymphatic drainage, which was directed to the homolateral axillary region, to the lymphoid tissue of the upper mediastinum and to the anterior region of the neck began earlier . Thoracic focalisations were present for all the patients, whereas bowel focalisations were only observed for the subcutaneous route of administration . Sublingual SIT did not induce axillary or intestinal' inflammatory focalisations, even though the patients had swallowed the allergenic extract . The uptake coefficient in individualized areas corrected to the uptake coefficient background was also studied .
Conclusions : For the subcutaneous route of administration, except for gluteraldheyde modified allergen, the local inflam-matory activity at the allergenic injection site was significantly higher'indepth and was time dependent, maintaining activity even after complete disappearance of the erythema and/or wheat These results express a prompt inflammatory involvement of the immune system with this allergenic therapy, which was unexpected until now . We also observed differences concer-ning allergic diseases, the type of allergenic extracts and routes of administration .
Keywords: Specific immunotherapy Subcutaneous immunotherapy Sublingual immunotherapy Allergic inflammation -Radio labelled cells - Scintigraphy, s mTc-HMPAO .
INTRODUCTION
P
llergen specific immunotherapy (SIT) and total avoidance of exposure to allergens, to which the patient is sensitised, are the only interven-tions that really influence in the long-term control of the allergic disease (1, 2) .
(1) Immunoallergology Department, Coimbra University Hospital ; Pneumology Center - Coimbra University (Portugal)
(2) Biophysical Department, IBILI, Medicine Faculty - Coimbra University.
(3) Nuclear Medicine Department - Coimbra University Hospital . (4) Medicine Faculty of Lisbon University .
European Annals of Allergy and Clinical Immunology - volume 36 - n° 10 - 2004
In fact, it is now obvious that SIT is the only curative treatment for hymenoptera venom allergy or latex anaphylaxis, two examples of life-threatening IgE dependent mechanisms (1-4) . A lot of DBPC studies (double-blind, placebo-controlled) provide longitudi-nal data on the clinical efficacy of subcutaneous SIT on mite, pollen, epithelium and some mould species . For other respiratory allergic diseases such as bron-chial asthma, allergic rhinitis and conjunctivitis, the reduction of symptom scores/need for medication were also observed (5) .
Allergen vaccines are, in fact, the only treatment that may affect the natural course of allergic disease, pre-venting the development of asthma in patients with allergic rhinitis, reducing the severity of the disease
and the need for anti-allergic drugs thereby impro-ving the patient's quality of life (1) . It is also now reco-gnized that immunotherapy is long-lasting effective and should be started earlier in the course of the disease . It has been propose that SIT reduce the risk of development of new sensitisations (6, 7), but recent data has not confirmed this protective effect on monosensitised allergic patients (8) .
There are several factors that are important to the cli-nical outcome of SIT concerning efficacy and toleran-ce : the use of premedication, the allergen sourtoleran-ce, the purification, the standardization and the stability of the extract, the pharmaceutical presentation of the allergen (native or chemically modified ; aqueous or depot) . The clinical manifestations before and during the treatment, the route of SIT administration, and the
schedule followed, should also be evaluated (9) . The aqueous extracts are highly effective, but induce more side-effects (local and systemic) than depot and modified vaccines . These have been developed in an attempt to reduce or remove allergenicity, while pre-serving or increasing the immunogenicity. Aqueous subcutaneous SIT is now the gold standard treatment for hymenoptera venom allergy and depot aeroaller-gen extracts for respiratory allergy . The use of chemi-cally modified allergens is not consensual . Although recent papers demonstrate a clinical benefit with aller-goid therapy, DBPC studies comparing both extracts are still missing (10) .
Local immunotherapy is another strategy for allergic disease treatment . In spite of the therapeutical bene-fit observed in some studies, there is no consensus on the use of the nasal, bronchial or oral route of admi-nistration of SIT (1, 2, 11) . On the other hand, the sub-lingual-swallowed administration of SIT in higher doses, which is another type of local immunotherapy, has an A level evidence-based medicine for seasonal and perennial allergic rhinitis in adults and seasonal allergic rhinitis in children (1, 11, 12) .
The clinical efficacy was demonstrated by DBPC study and later, with the increment in the allergen doses, the immunological changes were confirmed (12, 13) . Recent papers have confirmed the long-lasting (14) clinical efficacy of sublingual administration in a ran-domised, placebo-controlled, double blind, double-dummy study (15) .
In spite of the widespread knowledge in the scientific literature, the mechanism by which SIT achieves cli-nical improvement remains unclear . For subcuta-neous immunotherapy the immunomodulation of the T-cell response is now accepted : immune deviation (increase in ThO/Th1), Tcell anergy (decrease in Th2/ThO), or both mechanisms (1) . However, this is
not so linear, because SIT did not induce Th1-type cytokine-associated pathology in treated respiratory allergic patients to aeroallergens (16) . There are other
3 76
immunological changes with subcutaneous SIT : the rise in allergen-blocking IgG, the induction of IgE-modulating CD8+ T cells, the reduction of mast cells and eosinophils on the target mucosa, the decrease in inflammatory mediators (1, 5, 17-21) and a modula-tion on the inflammatory trafficking cells by reduced expression on VCAM-1 (22, 23) .
Immune tolerance depends on different mechanisms, including Tcell anergy, Tcell depletion by apoptosis, and active immune suppression (24) . One of the target mechanisms of SIT is the induction of tolerance to aller-gens to which the patient is sensitised (25) . IL-10 is pro-bably a relevant cytokine induced by this treatment and is related to regulatory T cells that actively control or suppress the function of others cells, generally in an inhibitory pattern (5, 25) . The changes in microenviron-ment due to the decrease in histamine and PGE2 mast cell release and the IL-10 and TGF-_ release by dendritic cells could deviate the Tcell population pathway in order to induce T regs and finally a therapeutic toleran-ce (26) . This retoleran-cent findings agree with what is current-ly known about the regulation induced by SIT . It is not credible that it could promote a Th2 / Thl switch (25) . Much of the knowledge on SIT is based on studies performed in subcutaneous route of administration, but increasing data are now available on sublingual vaccines . The absorption of allergen through the intact mucosa and the interaction with local dendritic cells could induce the process and immune-tolerance . Besides the intrinsic local effect, the swallowed aller-gen could induce an additional GALT related mecha-nism (11, 12) . In fact, the first method of sublingual administration of SIT in which the allergen was spat out is significantly less effective . Most of the mecha-nisms and immunological changes that occur with sublingual administration of high allergen dose have been recently demonstrated (1, 27-31) .
There is lots of information available on immunomo-dulation with SIT, but studies on kinetics of SIT admi-nistration and its immune effects on allergic patients are still lacking .
The study of M Bagnasco et al. (32) was highly impor-tant in understanding much of the data . In this study, the administration of radiolabelled Par-j-1 by 123 1 isoto-pe to healthy individuals demonstrated the local isoto- persis-tence of the allergen at the sublingual region for a long period of time . The systemic radioactivity was only pre-sent after swallowing the allergen . For sublingual SIT it seems that the allergen is degraded in the gastroenteral environment and only peptides are absorbed (33) . There are no similar studies in the literature concerning the subcutaneous route of administration .
Radiolabelling of blood cells is a nuclear medicine technique used in the study of inflammatory diseases such as chronic bowel inflammatory disease (34-37) . We considered that this kind of methodology could be
interesting worth investigating further with regards to an in vivo approach to the study of SIT mechanism of action and immune response . We expected to study the migration of autologous peripheral labelled-leu-kocytes that were reinjected to the site of the allergen extract administration in allergic patients, and the other possible resulting events . We think that the bio-logical response to the therapy can be easily obser-ved . The site or sites where the immune response happen and the different times of the response, can be established . We decided to study the kinetic res-ponse to SIT in patients with different types of allergy concerning the severity, the type of allergenic extracts administered and different routes of administration .
MATERIAL AND METHODS
The Coimbra University Hospitals Ethics Committee and Research Commission approved the design of this study protocol and all the volunteer patients signed an informed consent form according to the international ethics consensus .Subjects
13 adult allergic volunteer patients aged 20 to 51 year old were enrolled for study . All of them were submit-ted to SIT for at least 2 years and in all of them the cli-nical efficacy was demonstrated by complete remis-sion of symptoms, the absence of medication with regular anti-allergic drugs, reduction of skin reactivity to allergens and reduction of serum specific IgE . In
patients with previous anaphylaxis, the absence of allergic reactivity after the beginning of treatment was confirmed by challenge tests or immunoblotting studies .
The patients were selected according to the different allergenic extracts and routes of administration of SIT,
including :
a Aqueous subcutaneous allergenic extract (for ana-phylaxis) :
• 2 patients - hymenoptera venom, honeybee venom extract ;
• 3 patients - latex extract .
- Depot subcutaneous allergenic extract (for respirato-ry allergy) :
• 2 patients - Dermatophagoides pteronyssinus extract ; • 2 patients - grass pollen extract .
Modified subcutaneous allergenic extract (for respi-ratory allergy) :
• 1 patient - grass pollen extract; • 1 patient - Parietaria judaica extract .
Sublingual allergenic extract (for respiratory allergy) : • 2 patients - Dermatophagoides pteronyssinus extract .
European Annals of Allergy and Clinical Immunology - volume 36 - n° 10 - 2004
Dermatophagoides pteronyssinus allergic patients (bronchial asthma and rhinitis) not submitted to SIT were studied as a control group .
None of the patients had other clinical disorders, namely inflammatory diseases . All the female patients were submitted to a pregnancy test on the day of the study to exclude pregnancy .
All the studies were performed under strict hospital medical care surveillance, in the day of SIT administra-tion, according to each individual schedule of treatment .
Methods
∎ Technetium -99m hexamethylpropyleneamineoxime leukocyte labelling (99mTC-HMPAO) : The white blood cells labelling process with 99mTC-HMPAO was per-formed according to the standard University Hospital Nuclear Medicine Department protocol, based on the technique described by Peters et al. (34) . 42 ml of the patient's blood was collected from peripheral vein with a 60 ml plastic syringe, containing 6 ml of acid-citrate-dextrose (ACD) plus 6 ml of hydroxy-ethil-star-ch (HES) at 6% .
The ACD anticoagulant prevents cells from sticking to the plastic wall of tubes and the HES accelerates ery-throcyte sedimentation by affecting the charge of the sialic acid groups on the outer membrane of the red blood cell . This blood in the syringe was maintained in a vertical position for 30 mins in order to allow ery-throcyte sedimentation . A second 10 ml syringe of blood was sampled with 1ml of ACD . This blood was centrifuged at 2 000 g for 10 min in order to obtain cell-free plasma (CFP) .
After 30 mins of blood syringe sedimentation, the supernatant was centrifuged at 150 g for 5 min and the supernatant, platelet rich plasma (PRP) was dischar-ged . The leukocyte pellet was resuspended in 0 .6 ml of cell free plasma and labeled with 99mTC-HMPAO (Ceretec®, Amersham, UK) . For the leukocyte labeling procedure, the HMPAO must be labeled first . For this purpose, a 1110 MBq in 5 ml of saline of 99mTc fresh-ly eluted was added to a kit of HMPAO that contained 0 .5 mg of exametazime, 7 .6 mg of SnCl 2 and 4 .5 mg of NaCl according to the manufacturer's indications. 3 ml of this 99mTc-HMPAO was added to the leukocytes resuspended in cell free plasma, and the mixture was maintained at room temperature for incubation, for 10 min, in order to label the leukocytes . After this time, 10 ml of PRP was added to the leukocytes in order to stop the labeling procedure, and the mixture was centrifu-ged at 150g for 5 min .
After centrifugation, the supernatant was removed and discharged and the labeled leukocyte pellet was resuspended in 5 ml of CFP, counted and re-injected into the patients .
The labeling yield ranged between 50% and 75%, as normally described .
The effective dose-equivalent for this examination was 0 .021 mSv/MBq (38, 39) .
•
Allergenic therapeutic extract : The allergenic extra-cts administered were from ALK-Abe116 (Spain) for all the patients except for modified subcutaneous aller-genic extract . These extracts were glutaraldehyde modified grass pollen (Bial/Aristegui- Spain) and depigmented Parietaria judaica pollen (Leti- Spain) . All the patients were submitted to the maintenance dose according to the individual schedule .
One allergic patient of the control group was submitted to a 0.5cc subcutaneous phenol aqueous injection sup-plied by ALK-Abe116 (Spain), and the other patient was submitted to a 0 .5cc subcutaneous injection of a bacte-rial extract (RibomunylTM, Pierre Fabre Medicament, France : ribosomal fractions of Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzaeand the membrane fraction of
Klebsiella pneumoniae), an nonspecific immunostimu-lant agent of IgG production .
• Administration procedures : Patients were lying
supine under the gamma camera . A humeral vein was catheterised to permit the reinjection of labeled auto-logous leukocytes . The allergen extract was adminis-tered subcutaneously, according to the usual method, in the contra lateral arm . Both injections were given at the same time, simultaneously with the scintigraphic acquisitions .
The 2 allergic patients under sublingual SIT were also lying supine under the gamma camera at the begin-ning of the study . The drops of the allergenic extract were administered at the same time as the reinjection of the labeled autologous leukocytes in the humeral vein . Three minutes later, the-patients swallowed the allergen extract .
For the control group the same procedure was adop-ted . The study was performed under the close super-vision of allergist and nuclear medicine specialists .
• Scintigraphic acquisitions (table 1) : Scintigraphic studies were performed on a large field of view digi-tal gamma camera (GE XR, Milwaukee, USA,
interfa-maximum count maximum count Uptake Coefficient
Uptake Coefficient
average count
average count background
max count ROI /average count backgrou ! max count /average countUptake COW UCROI - UCBK
Table 1 .
3 78
ced with a Camstar acquisition unit and an eNTEGRA processing unit), equipped with a parallel low-energy general purpose collimator .
A dynamic acquisition was obtained in anterior view at thoracic and neck level for 64x64 matrix for 60 minutes (120 frames x 30 seconds) followed by static images at 60, 90, 120, 180, 240, 300 and 360 mins after administration of the radiolabeled leukocytes and the allergen for 256x256 matrix and for 5 mins each . These static images were obtained in anterior and posterior views at thoracic level and anterior views at abdominal projection .
During the acquisitions, the visualized area was kept close to the gamma-camera collimator, and the patients were not allowed to move any part of the body, in order to maintain the geometry of the projections .
Throughout the acquisitions, the erythema and the wheal at the site of SIT administration were controlled . For quantitative results, the procedure was different according to the type of image . For the dynamic tho-racic images, regions of interest (ROls) were drawn at SIT administration, background area (muscle), and for possible focalisations (cervical and axillary areas and thorax) . For the thoracic static views, an image corresponding to the geometric mean bet-ween anterior and posterior views was calculated, and ROls were drawn at thoracic locations . For the static anterior abdominal views, ROls were drawn over each location .
For each ROI the total counts, average counts per pixel and maximum counts were obtained, and decayed was corrected . With these values, uptake coefficients were calculated by the ratio between the maximum count rate at the target ROI and average counts at the background ROI . In order to normalise and to help with the interpretation of the results an uptake coefficient of the background ROI was calcu-lated and subtracted from the uptake coefficient of the target ROls .
∎ Data analysis : The clinical pattern, allergen extra-ct and route of administration were analysed indivi-dually and the different groups of patients were compared .
The statistical analysis was performed by descripti-ve analysis .
alts
There were no adverse events caused by radiolabeled autologous cells reinjection or allergenic therapeutic extract administration .
Clinical and allergenic therapeutic extract aspects are shown in table 2 and 3 .
After scintigraphic processing, the ROI value was obtained at the site of SIT administration, thoracic, axillary and cervical area, abdominal focalizations and ROI background . We also calculated the corrected uptake coefficient (maximum count at the site of SIT ROI over average counts at the background ROI) at different focalisations at different times .
The uptake coefficients for the different focalisations on analysis are shown in Figs . 1-5 .
We did not consider the scintigraphic acquisitions at the site of the allergenic injection (patient 1, 2 and 9) and the axillary area (patient 1) due to technical pro-blems .
The inflammatory activity started early in patients treated with aqueous allergenic extract for anaphy-laxis than for in patients treated with depot extracts
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European Annals of Allergy and Clinical Immunology - volume 36 - n° 10 - 2004
for other diseases (latex : 20-35 mins; hymenoptera venom : 15 mins ; grass pollen : 25 and 30 mins; mites: 25 and 45 mins) .
For sublingual allergenic extracts the inflammatory activity started promptly (at 5 mins) with a rapid increase that persisted during the study .
In patients submitted to allergoid, the inflammatory activity at site of administration of the extract started in the first minutes, although with a different pattern . In the allergic patient submitted to saline subcuta-neous injection, we observed low values of activity at the site of administration, probably related with an irritative effect caused by phenol used as preservati-ve . This observation confirms the sensitivity of the laboratory procedure . No other focalisations were observed during the study .
The control patient submitted to bacterial injection sho-wed inflammatory activity at 10 minutes with an increa-se that was time related . We also obincrea-served activity in the axillary, the cervical and the thoracic areas, but with
no focalisations at the bowel region, Fig . 6.
The inflammatory activity at the axillary and the cer-vical areas was present from the beginning of the
Figures 1 : Uptake coefficient corrected o allergenic extract local administration .
Figures 2 : Uptake coefficient corrected axillary area focalisations .
I
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Table 2 : Clinical aspects of patients sample .
Disease : A= anaphylaxis ; BA+AR= bronchial asthma+allergic rhinitis .
Allergen : L=latex ; HV= hymenoptera venom (honeybee) ; GP= grass pollen ; Dp= Dermatophagoides pteronyssinus judaica
SIT route : sc= subcutaneous ; sl= sublingual
SIT extract : depot= aluminium hydroxide-adsorbed ; alergoide=
SIT (cc) : volume in ml administrated of the maintenance dose . Patients 12 and 13 are submitted to 5 drops SIT dose: quantity of allergen in µg protein
Administered activity : MBq
Table 3 : Control group . Clinical aspects
Disease : BA+AR= bronchial asthma+allergic rhinitis . Allergen : Dp= Dermatophagoides pteronyssinus Administered activity : MBq
arietaria
Controls
Sex
Age
(y)
Disease
Evolution (y)
Disease
Allergen Injection Volume(cc) Administered
activity
1
F
41
20
BA+AR
Dp
saline
0 .50
306 .4
2
F
45
20
BA+AR
Dp
bacterial
0 .50
126 .0
Patient
Sex
Age
(y)
Disease
Evolution (y)
Disease
Allergen
SIT
route
SIT
extract
SIT
years
SIT
(cc)
SIT
dose
Administered
activity
1
F
34
7
A
L
sc
6
0 .35
0 .35
272 .0
2
F
39
6
A
L
sc
5
0 .35
0 .35
240 .5
3
F
28
7
A
L
sc
5
0 .35
0 .35
149 .6
4
M
49
5
A
HV
sc
4
1 .0
100
67 .7
5
F
37
4
A
HV
sc
4
1 .0
100
316 .3
6
F
53
14
BA+AR
GP
sc
depot
4
0 .60
1 .5
337 .2
7
F
24
10
BA+AR
GP
sc
depot
4
0 .60
1 .5
255 .5
8
M
22
12
BA+AR
Dp
sc
depot
4
0 .60
3 .6
91 .8
9
F
51
30
BA+AR
Dp
sc
depot
5
0 .60
3 .6
170 .3
10
M
33
28
BA+AR
GP
sc
allergoide
5
0 .70
36 .72
476 .7
11
F
49
34
BA+AR
Pj
sc
allergoide
3
0 .50
12 .0
132 .7
12
M
20
10
BA+AR
Dp
sl
aqueous
3
5*
1 .2
126 .3
13
F
27
19
BA+AR
Dp
sl
aqueous
3
5*
1 .2
254 .6
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acquisitions for the subcutaneous allergenic extracts . There was a stabilization of the uptake coefficient values at 180 minutes . There were no focalisations at the axillary areas after sublingual immunotherapy . The high-labelled cell circulating pool could affect the
interpretation of focalisations in the lung area in the first minutes, so we decided to calculate ROls only
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European Annals of Allergy and Clinical immunology - volume 36 - n° 10 - 2004
Figures 3 : Uptake coefficient corrected cervical area focalisations .
igures 4 : Uptake coefficient corrected lung focalisations .
Figures 5: Uptake coefficient corrected o gut focalisations .
after 60 minutes after the injection, even though they were observed earlier . There were no significant dif-ferences among the different groups .
Focalisations at the abdomen acquisition views were observed in all the patients submitted to subcuta-neous SIT. We only considered ROls till 240 minutes . This time was coincident with the beginning of
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tic/gall bladder excretion of the product. No bowel acti-vity was observed in patients on SLIT treatment . There were other focalisations such as supraster-num and anterior mediastisupraster-num with high inflamma-tory activity, but we decided not to consider these areas because they were sites of vascular confluen-ce . Bone marrow was another site of intense
activi-3 8 2
Figures 6 : Uptake coefficient corrected/time for the ROls of the active control patient sub-mitted to bacterial extract .
Figures 7 : Patient 1 study, dynamic and sta-tic acquiitions . Inflammatory activity at 25, 90 120, 180, 240 min .) .
Figures 8 : Patient 12 study, dynamic and static acquisitions . Inflammatory activity at 240 min .
ty, higher than that usually observed by this tech-nique .
Distinct ROI count profiles were obtained with the dif-ferent routes of administration and with the difdif-ferent allergenic extracts but all of them induced a time-dependent global inflammatory activation at sites related with the immune system (Fig . 7-10) .
European Annals of Allergy and Clinical immunology - volume 36 - n° 10 - 2004
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4?
G acgLftS t
Concerning depot allergenic extracts, we did not observe differences between pollen and mite allergic patients .
For mite allergic patients, there were the same obvious differences concerning SIT routes of extract administration . The sublingual immuno-therapy induced an early potent activity with higher uptake coefficient ROI values than the depot extracts, at site of the allergic extract
admi-nistration .
The inflammatory activity at the local of SIT admi-nistration was greater than the erythema area involving the deeper layers and persisted even after local inflammatory signs disappeared .
European Annals of Allergy and Clinical immunology - volume 36 - n° 10 - 2004
DISCUSSION
In this study we decided not to label the allergen used, because it was only possible to make a linkage between an allergenic epitope and the radiolabel-drug, excluding the other epitopes . The decision to use the common administration of SIT seemed to be the procedure that minimised the error factors in the understanding of this therapy dynamics . The technique is now a routine pro-cedure for analogous inflammatory pathologies . The risks are minimal, the radioactive burden is low, and the body-absorbed dose (effective dose) corresponds to about 3mSv, which is similar to one year of natural exposure in the continental area of Portugal .
Our first attempt was to study the local inflammation related to SIT administration . The activity observed in other areas and the speed of occurrence was a sur-prising result. Even in patients with excellent clinical response to immunotherapy, the administration of specific allergen resulted in a local inflammatory res-ponse that ranged from minimal intensity to erythe-ma and/or wheal . The intensity of inflamerythe-mation was higher for aqueous extract administrations in patients with the most severe allergic diseases . However, it was not related with clinical signs of inflammation at the site of administration . The allergoids were related with lower inflammatory activity when compared with depot extracts, as expected, according to the cli-nical knowledge (40) . In the 2 patients submitted to sublingual extracts the beginning of the radioactivity was faster, with no signs of local effects . There was a minimal activity in the control patient who was sub-mitted to phenol-saline injection, which is usually used to dilute the extracts . We considered that this reaction was related to phenol, which is a nonspecific irritant used as a preservative . This observation confirmed the sensitivity of the technique . The other control patient that was submitted to an injection of bacterial derived fragments, also revealed activity according to the expected IgG dependent mechanism of action of this immune-stimulant (41) .
In the first minutes ascending drainage directed to the homolateral axillary area was induced by subcuta-neous SIT. Cervical and superior mediastinum lym-phoid tissues were the next structures involved . The late static acquisitions showed global activation on focalisations related to the immune system : lymphoid tissue neck/cervical, suprasternum, mediastinum, lung, gut and bone marrow. The early involvement of the central lymphoid structures was an unexpected finding . The maximum ROI values were increased even when the radionuclide was eliminated through urine and the half time was reached . This could reflect the magnitude of the therapeutical allergen mecha-nism of restimulation and immune-tolerance .
Concerning the sublingual immunotherapy the activi-ty was reached faster than for subcutaneous SIT. There was an obvious difference in the inflammatory activity profile comparing these two types of immu-notherapy. The most intense ROI values were achie-ved at the cervical lymphoid tissue, as expected . There was no activity in the axillar and the abdominal area, compared with depot extract . This data could explain the need for daily or three times a week admi-nistration of sublingual SIT in order to promote fre-quent stimulation of the immune system .
The allergoid extracts also induced inflammatory acti-vity when compared with depot pollen extracts . There were obvious differences in local activity between both . Our results could deflect some criticism of lot of
3 8 4
authors who reject the use of polymerised vaccines or chemically modified allergens .
This technique did not allow labelling specific circula-ting peripheral cells . The pellet resulcircula-ting from a blood sample contains all the lineages of white blood cells. At first, the lymphocytes seemed to be the only cells responsible for the local and global events in the study . However, we must reflect on all the knowledge about SIT, inflammation and immune response avai-lable till now .
Dendritc cells (DC) have been a matter of exhaustive study and seem to be a nuclear target of inflammation and immune tolerance . The professional APC are divi-ded into two major systems : DC, including blood and tissue DC as well as epidermal Langerhans cells (LC),
and the monocyte/macrophage system (42) .
Concerning DC there are two different subsets : DC1 or myeloid DC, and DC2 or lymphoid/plasmocytoid DC (42, 43) . DC2 are mainly found in thymic medulla, ton-sils and in T cell areas of secondary lymphoid organs, whereas DC1 are located outside the T cell areas . DC2 are unable to internalise foreign antigen (42) . On the other hand, DC1 could traffic from the blood to the peripheral tissue to capture foreign antigens, may migrate to the draining lymphoid organs in order to prime T cells towards Th1 or Th2 cytokine profile (26, 43, 44) . Under appropriate stimulus peripheral blood monocytes could differentiate to DC1 (42) .
Langerhans cells belong to the DC1 system and are relevant in the skin . After local inflammatory stimulus the monocyte-chemoattractant protein (MCP) induces recruitment of LC progenitors from the bone marrow, migration to the skin and in the skin, and migration to peripheral lymph nodes (43) . It is now well established that IL-10 DC release is an important factor of immune tolerance promoting Tregs lymphocytes (25, 26, 45) . The DC family of oral mucosa is different from that of the skin LC family. The oral microenvironment and
TGF-R
are relevant as well as IL-10 (43) . They also presented increased expression of FccRl on their surface, which was partially occupied by IgE molecules (43) . The linka-ge of allerlinka-gens to the FcERI receptor contributes to tole-rance by an IL-10 dependent mechanism (42, 43) . According to our results these data could be explai-ned by the following hypothesis : The subcutaneous allergenic extract induced activation of LC, recruit-ment of other circulating DC/monocytes ; migration to lymphoid areas ; cooperation with DC2 and finally Tregs induced by IL-10 and tolerance towards sensiti-sed allergens . The systemic activation of areas related with the immune system could support the global imunomodulation of SIT.Large amounts of allergen are administered by sub-lingual SIT route in an area with close proximity to lymphoid organs such as the lingual amygdale . It is possible that DC could initiate the process as occurs
saturation of activity of DC1 and the inability to cause an intense systemic inflammatory activity . The indu-ced tolerance and the efficacy of this therapy are, consequently less intense and require frequent stimu-lation and higher allergen doses . On the other hand, the swallowed allergen could interact with gastroin-testinal mucosa and promote additional tolerance mechanism, mediated by TGF-(3 and Th3 regulatory cells (43, 45) .
Our results showed a real systemic effect of SIT, rela-ted to focalisations on immune system dependence . The inflammatory activity began quicker than we expected and there were obvious differences concer-ning the different extracts . There seems to exist an obvious difference between the kinetic of sublingual and subcutaneous routes of administration . Aqueous allergenic extracts induce more intense and global systemic inflammatory activity than depot extracts and ultimately the allergoid extracts .
In the future it will be important to study the kine-tic/dynamic of the built-phase of SIT in animal models in order to understand how the tolerance/immuno-modulation process occur throughout the treatment .
Acknowledgments :
We thank Drs I . Carrapatoso and G . Loureiro for helping with the clinical study ; Nuclear Medicine technicians ; and Jeremy Waterson for the English revision . Research Grant from Pneumology Center of Coimbra University (Dir . Prof . L .-C . Oliveira)/Portuguese Ministry of Education .References
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AVOIDING PEANUTS MAY RESULT IN RECURRENCE OF ALLERGY. FROM THE
JOURNAL OF ALLERGY & CLINICAL IMMUNOLOGY
Children who keep avoiding peanuts after they outgrow their peanut
allergy -h
3
rat
their
peanut allergy will return, according to a study in the November 2004 Journal ;3f_Alle
I 'Immunology (JACI) . The JACI is the peer-reviewed journal of the American Academy ofAI11
*>k &'Immunology (AAAAI) .,study,
David M .
Fliescher, MD, Johns Hopkins University School of Medicine, andcolleagi
e .ants
who
had previously outgrown their peanut allergy . They sought to determine thepercefda
who
reacquire peanut allergy later; identify any risk factors for recurrent peanut allergy, and'de
c -
+'eeE emendationsfor treating patients who have outgrown peanut allergy.on
dtlestionnaire results, repeat peanut-specific IgE levels, and repeat food challenges, thereseer6ti' that
patients who have outgrown their peanut allergy have approximately an 8% chance of ftr
'The
studyfound
this risk is significantly higher in patients who continue to avoid eating peanUtafter-is resolved .
n the study's results, researchers recommend that patients eat concentrated forms of'penutof 1 cinchafter outgrowing their peanut allergy in an attempt to maintain their tolerance topeanu
e
ers:
also recommended that patients and families who rarely eat peanuts or eat then~4n f"it i'4 meroutgrowing their allergyshould continue to
carry epinephrine at all times .I is the largest professional medical specialty organization in the United States representing
allergists, ast
' ;
atti&ts,iclinical immunologists, allied health professionals and others with a special interest in the reseami
and w_ ,
of&Ifergicdisease . Established in 1943, the AAAAI has nearly 6,000 members in the United States,Canada
ar+d `tries .
The
AAAAI serves as an advocate to the public by providing educational information throughits Web l iatY .',saaai,orgor the toll-free physician referral and information line at 1(800) 822-2762 .
ffIRIS NOTE: This study was published in the peer-reviewed, scientific journal of the American Academy: of Alte
& immunology, but does not necessarily reflect the policies or the opinions of the Academy . To receive
a yL
sleasl~contact John Gardner ( j gardner@aaaai .or g ) at (414) 272-6071 . For more information and mess tiA
past JACI news releases, visit the Media Center of the AAAAI Web, site, www .aaaai .org .
